Authors:A. Robic; K. Feve; J. Riquet; A. PrunierPages: 1 - 9Abstract: Publication date: Available online 9 April 2016 Source:Domestic Animal Endocrinology Author(s): A. Robic, K. Feve, J. Riquet, A. Prunier The present study was performed to measure mRNA levels of steroidogenic enzymes in testes and fat tissue and determine whether they are related to fat androstenone level. Real time PCR experiments were performed on 26 testes and 12 adipose tissue samples from pubertal boars using 21 genes. The absence of significant correlations between fat androstenone and the transcriptional activity of the SRD5A2 and SRD5A3 genes but the high correlation coefficient with that of the SRD5A1 gene (r = 0.62, P < 0.05) suggest that the enzyme coded by SRD5A1 is mainly responsible for the last step of androstenone synthesis. The testicular transcriptional activities of CYP17, CYP11A1, CYP19A, AKR1C-pig6, SRD5A1, LHCGR, and AR were significantly correlated. Only transcriptional levels of CYP17, CYP11A1, CYP19A, SRD5A1 and AKR1C-pig6 were correlated with the fat concentration of androstenone (0.57 < r < 0.70, P < 0.05) confirming that the amount of androstenone stored in fat is related to the production in testes of androstenone and more generally to all sex steroids. Altogether, our data are in favor of a preponderant role of AKR1C-pig6 instead of HSD17B3 for testicular synthesis of steroids. Concerning fat tissue, our data do not support a significant de novo biosynthesis of steroids in porcine adipose tissues. The presence of transcripts coding for steroid enzymes, especially those of AKR1C-pig6, suggests that steroids can be transformed. None of transcript abundance was related to androstenone accumulation (P > 0.1). Therefore, steroids synthesized elsewhere can be transformed in fat tissue but synthesis of androstenone is unlikely.

Authors:K. Kusama; R. Bai; T. Sakurai; H. Bai; A. Ideta; Y. Aoyagi; K. ImakawaPages: 21 - 30Abstract: Publication date: October 2016 Source:Domestic Animal Endocrinology, Volume 57 Author(s): K. Kusama, R. Bai, T. Sakurai, H. Bai, A. Ideta, Y. Aoyagi, K. Imakawa Interferon tau (IFNT) is the pregnancy recognition protein in all ruminants, and its expression is restricted to trophoblast cells. Interferon tau production increases as the conceptus elongates; however, its expression is downregulated soon after the initiation of conceptus attachment to the uterine epithelium. Our previous study identified that among 8 bovine IFNT genes, only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the periattachment period. To characterize whether Hippo signaling including a transcription cofactor yes-associated protein (YAP) was involved in the IFNT regulation, we examined the expression and effects of YAP and/or TEAD in human choriocarcinoma JEG3 and bovine trophoblast CT-1 cells, and in bovine conceptuses obtained from day 17, 20 or 22 pregnant animals (pregnant day 19.5 = day of conceptus attachment to the endometrium). YAP was expressed in bovine conceptuses and transfection of YAP or TEAD4, a transcription factor partner of YAP, expression plasmid increased the luciferase activity of IFNT2 and IFN-tau-c1 reporter plasmids in JEG3 cells. In the presence of YAP expression plasmid, TEAD2 or TEAD4 expression plasmid further upregulated transcriptional activity of IFNT2 or IFN-tau-c1 constructs, which were substantially reduced in the absence of the TEAD-binding site on IFNT2 or IFN-tau-c1 promoter region in JEG3 cells. In CT-1 cells, treatment with TEAD2, TEAD4, or YAP small-interfering RNA downregulated endogenous IFNT expression. It should be noted that TEAD2 and TEAD4 were predominantly localized in the nuclei of trophectoderm of Day 17 conceptuses, but nuclear localization appeared to be lower in those cells of conceptuses on days 20 and 22 of pregnancy. Moreover, the binding of TEAD4 to the TEAD-binding site of the IFN-tau-c1 promoter region in day 17 conceptuses was less in day 20 and 22 conceptuses. Furthermore, the level of YAP phosphorylation increased in day 20 and 22 conceptuses. These results indicated that although YAP/TEAD had the ability to up-regulate IFNT gene transcription on day 17, IFNT2 or IFN-tau-c1 was down-regulated following changes in the localization of TEAD2 and TEAD4 from the nucleus to the cytoplasm and increases in phosphorylation and degradation of YAP. These data suggest that TEAD relocation and/or YAP degradation following its phosphorylation down-regulates IFNT gene transcription after conceptus attachment to the uterine endometrium.

Authors:M.L. Cobb; K. Iskandarani; V.M. Chinchilli; N.A. DreschelPages: 31 - 42Abstract: Publication date: October 2016 Source:Domestic Animal Endocrinology, Volume 57 Author(s): M.L. Cobb, K. Iskandarani, V.M. Chinchilli, N.A. Dreschel Salivary cortisol is widely used as an indicator of stress and welfare in canine research. However, much remains unclear about the basic features of this hormone marker in domestic dogs. This systematic review and meta-analysis aimed to determine a reference range for cortisol concentration in the saliva of dogs and examine how canine characteristics, environmental effects and experimental considerations relate to salivary cortisol concentrations. A systematic review of literature databases and conference proceedings from 1992 to 2012 identified 61 peer-reviewed studies using domestic dog salivary cortisol. Researchers were contacted via email, and 31 raw data sets representing a total of 5,153 samples from 1,205 dogs were shared. Meta-analysis provided a cortisol concentration range of 0 to 33.79 μg/dL (mean 0.45 μg/dL, SEM 0.13). Significant effects (P < 0.05) were found for sex and neuter status, age, regular living environment, time in environment before testing, testing environment, owner presence during testing, and collection media. Significant effects were not found for dog breed, body weight, dog type, coat color, assay type, exercise, eating, or use of salivary stimulant. Care should be taken when using cortisol studies for dogs at a group or population level as there is a large amount of intraindividual and interindividual variability and external variables could influence salivary cortisol concentration. This analysis highlights the importance of carefully controlling experimental design to compare samples within and between individual dogs, as well as establishing and using best practices for saliva collection. Caution should be exercised in comparing different studies, as the results could be the reflection of a plethora of factors.

Authors:M.D. Scheidegger; V. Gerber; A. Ramseyer; G. Schüpbach-Regula; R.M. Bruckmaier; J.H. van der KolkPages: 43 - 47Abstract: Publication date: October 2016 Source:Domestic Animal Endocrinology, Volume 57 Author(s): M.D. Scheidegger, V. Gerber, A. Ramseyer, G. Schüpbach-Regula, R.M. Bruckmaier, J.H. van der Kolk The aim of this study was to further characterize the ACTH stimulation test as reflected by salivary cortisol response and to measure the short- and long-term repeatability of it in healthy horses as a tool to assess the capacity of the adrenal cortex to secrete cortisol. Nineteen healthy horses were subjected to 3 ACTH stimulation tests. Intervals were 2 wk and 5 mo between the first and second and the second and third tests, respectively. A dose of 1-μg/kg BW synthetic ACTH was injected intravenously. Saliva samples were collected at baseline and at 30, 60, 90, 120, 150, and 180 min after administration for cortisol measurements using a competitive enzyme immunoassay. A repeated measures ANOVA was used to compare values within and among horses. Mean ± SD total increase in cortisol concentrations integrated over the entire sampling period was 34.5 ± 11.0 ng/mL. The highest measured concentration at a single time point was 9.7 ± 2.7 ng/mL and was reached after 122 ± 22 min. For the short- and long-term repeatability, intraclass correlation coefficient was 0.90 and 0.33, respectively. The 3 ACTH stimulation tests results differed significantly among (P < 0.00001) but not within (P = 0.538) individual horses. The Freiberger stallions had a higher salivary cortisol baseline concentration and a lower response to ACTH stimulation as compared with Warmblood mares and geldings. The present study confirmed that the administration of ACTH in healthy horses reliably stimulates the salivary secretion of cortisol and shows that the test is repeatable in the short- and long-term.

Authors:I. Małysz-Cymborska; A. AndronowskaPages: 48 - 54Abstract: Publication date: October 2016 Source:Domestic Animal Endocrinology, Volume 57 Author(s): I. Małysz-Cymborska, A. Andronowska The influence of induction of ovulation and superovulation with eCG and hCG on LH and FSH receptor levels in porcine oviducts on day 3 postcoitum was studied. In experiment I, gilts were assigned into cyclic (control; n = 5) and inseminated (n = 5) groups. In experiment II, there were 3 groups of animals: inseminated (n = 5), induced ovulation/inseminated (750 IU eCG, 500 IU hCG; n = 5) and superovulated/inseminated (1500 IU eCG, 1000 IU hCG; n = 5) gilts. Oviduct tissues were collected 3 d after insemination or PBS infusion. The messenger RNA (mRNA) expression of FSH receptor (FSHR) and luteinizing hormone/chorionic gonadotropin receptor (LH/CGR) was measured by real-time reverse transcription PCR and protein levels using Western blots. Localization of LH/CGR and FSHR-positive cells was studied by immunohistochemical staining. Insemination by itself did not influence mRNA and protein levels of LH/CGR. However, FSHR mRNA expression in the isthmus and ampulla of the oviduct was affected by insemination (P < 0.05). Similarly, insemination decreased FSHR protein level in the isthmus (P < 0.05). Stimulation with hCG and eCG did not affect LH/CGR and FSHR mRNA expression, either in the isthmus or in the ampulla. Nevertheless, superovulation decreased LH/CGR protein level in the oviductal ampulla (P < 0.05) in comparison with inseminated gilts. Similarly, protein levels of FSHR in the oviductal ampulla decreased after superovulation (P < 0.05). LH/CGR-positive cells were observed in the mucosa as well as in smooth muscle cells of both parts of the oviduct. Follicle-stimulating hormone receptor–positive cells were observed in smooth muscle cells and blood vessels of the isthmus. In the ampulla, FSHR-positive cells were observed in the smooth muscle as well as in the mucosa. Summarizing, the present study revealed for the first time that stimulation with eCG and hCG, especially in high doses, can change LH/CGR and FSHR levels in porcine oviducts. This may in turn alter many signaling pathways, eg, PGs or vascular endothelial growth factor synthesis, and consequently disturb the oviductal environment, with possible detrimental effects on fertilization and/or embryonic development.

Authors:M.K. Reeve-Johnson; J.S. Rand; D. Vankan; S.T. Anderson; R. Marshall; J.M. MortonPages: 55 - 62Abstract: Publication date: October 2016 Source:Domestic Animal Endocrinology, Volume 57 Author(s): M.K. Reeve-Johnson, J.S. Rand, D. Vankan, S.T. Anderson, R. Marshall, J.M. Morton Diabetes is typically diagnosed in cats once clinical signs are evident. Diagnostic criteria for prediabetes in cats have not been defined. The objective of the study was to establish methodology and cut points for fasting and 2-h blood glucose concentrations in healthy client-owned senior cats (≥8 yr) using ear/paw samples and a portable glucose meter calibrated for feline blood. Of the 78 cats, 27 were ideal (body condition score [BCS] 4 or 5 of 9), 31 overweight (BCS 6 or 7), and 20 obese (BCS 8 or 9); 19 were Burmese and 59 non-Burmese. After an 18–24-h fast and an ear/paw blood glucose measurement using a portable glucose meter, glucose (0.5 g/kg bodyweight) was administered intravenous and blood glucose measured at 2 min and 2 h. Cut points for fasting and 2-h glucose concentrations were defined as the upper limits of 95% reference intervals using cats with BCS 4 or 5. The upper cut point for fasting glucose was 6.5 mmol/L. Of the overweight and obese cats, 1 (BCS 7) was above this cut point indicating evidence of impaired fasting glucose. The cut point for 2-h glucose was 9.8 mmol/L. A total of 7 cats (4 with BCS 8 or 9 including 1 Burmese; 3 with BCS 6 or 7, non-Burmese) were above this cut point and thus had evidence of impaired glucose tolerance. In conclusion, the methodology and cutpoints for diagnosis of prediabetes are defined for use in healthy cats 8 yr and older with a range of BCSs.

Authors:J.M. Baldrighi; M.A.R. Siddiqui; O.J. GintherPages: 80 - 84Abstract: Publication date: October 2016 Source:Domestic Animal Endocrinology, Volume 57 Author(s): J.M. Baldrighi, M.A.R. Siddiqui, O.J. Ginther The number and day of emergence (first detection) of 2-mm follicles and the number and day when the 2-mm follicles reached 3-, 4-, 5-, and 6-mm during wave 1 were determined every 0.5 d (n = 9 heifers). Emergence of the follicles at each of the indicated diameters was normalized to the beginning and ending nadir and the peak of each of a minor FSH surge, the preovulatory surge, and the periovulatory surge. Relative to the day of ovulation (day 0), the minor FSH surge, preovulatory surge, and periovulatory surge encompassed (nadir to nadir) days −7.0 to −2.5 (peak, day −4.0), days −2.5 to −0.5 (peak, day −1.0), and days −0.5 to 4 (peak, day 0), respectively. Distinct mean nadirs occurred between the minor and preovulatory surges and between the preovulatory and periovulatory surges. A small percentage of 2-mm follicles (12%) and 3-mm follicles (2%) emerged during the minor FSH surge. The 4-mm follicles emerged during the preovulatory surge (24% of follicles) and periovulatory surge (76%). The 5-mm and 6-mm follicles emerged only during the periovulatory surge. The first increase (P < 0.05) in number of 2-, 3-, and 4-mm follicles began at 1.5, 1.0, and 0 d, respectively, before the nadir at the beginning of the preovulatory surge. The first increase (P < 0.05) in number of 5- and 6-mm follicles began at 0.5 and 0 d, respectively, before the intervening nadir between the preovulatory and periovulatory surges. Results demonstrated that each of the 3 surges including the minor surge contributed to the emergence of follicles at various diameters during wave 1. The emergence of 2-mm follicles during the descending portion of the minor surge indicated that smaller follicles (eg, 1 mm) apparently emerged during the major portion of the minor surge. The increasing diameter of the 2 largest follicles was not interrupted during the distinct intervening nadir between the preovulatory and periovulatory FSH surges.

Authors:O.J. GintherPages: 85 - 99Abstract: Publication date: October 2016 Source:Domestic Animal Endocrinology, Volume 57 Author(s): O.J. Ginther Selection of the dominant follicle (DF) during a follicular wave is manifested by diameter deviation or continued growth rate of the largest follicle (F1) and decreased growth rate of the next largest follicle (F2) when F1 reaches about 8.5 mm in cattle. The process of deviation in the future DF begins about 12 h before diameter deviation and involves an F1 increase in granulosa LH receptors and estradiol and maintenance of intrafollicular free insulin-like growth factor 1 (IGF1). Thereby, only F1 is developmentally prepared to use the declining FSH in the wave-stimulating FSH surge and to respond to a transient increase in LH to become the DF. A follicle that emerges first may maintain an F1 ranking and become the DF by being first to reach a critical developmental stage. However, an early size advantage is not a requisite component of the deviation process as indicated by (1) F1 and F2 may switch diameter rankings during a common growth phase that precedes diameter deviation owing to intraovarian factors that affect growth of individual follicles; (2) any follicle that reaches 5 mm regardless of diameter ranking may become a DF unless it is selected against during deviation; (3) a subordinate follicle may become dominant if the DF is ablated; (4) when F1 is ablated at 8.5 mm, the next largest follicle that is greater than 7.0 mm or the first follicle to subsequently reach 7.0 mm becomes the DF; (5) after ablation of F1 at 8.5 mm, IGF1 and estradiol increase in the intrafollicular fluid of F2 beginning at 6 h, and F2 grows to 8.5 mm in 12 h to become the DF. These considerations indicate that selection of a DF or partitioning into a DF and subordinate follicles is not initiated before the end of the common growth phase. That is, the deviation process represents the entire follicle selection mechanism.

Authors:A.P. Foote; K.E. Hales; H.C. FreetlyPages: 100 - 107Abstract: Publication date: October 2016 Source:Domestic Animal Endocrinology, Volume 57 Author(s): A.P. Foote, K.E. Hales, H.C. Freetly Ghrelin is a peptide hormone produced in the gut that is implicated in signaling appetite and regulating dry matter intake (DMI). The objective of this experiment was to determine the change in acyl ghrelin, total ghrelin, and the ghrelin ratio (acyl ghrelin/total ghrelin) over an 84-d DMI and average daily BW gain (ADG) measurement period and to determine the association of those ghrelin measurements with DMI, ADG, ADG:DMI ratio (G:F), and residual feed intake in finishing beef steers and heifers. Blood samples were collected on day 0 and day 83 before feeding and between 0730 h and 1130 h. Samples were analyzed for acyl and total ghrelin using commercially available RIA. DMI in steers was greater during the last 35-d period of the experiment compared with the first 35 d (P < 0.01) and was greater than heifers regardless of period (P < 0.01). Steers had greater acyl ghrelin concentrations on day 0 than heifers, but concentrations decreased by day 83 to equal concentrations in heifers (P < 0.01). Total ghrelin concentrations were lower on day 0 in heifers but increased by day 83 and did not differ from steers on day 83 (P < 0.01). A mixed model analysis was used to determine the association of ghrelin concentrations and ratio with production traits, independent of breed and sire effects. There was an interaction of day 0 acyl ghrelin concentrations with time of sample collection for 84-d DMI (P < 0.01), ADG (P < 0.01), and G:F (P = 0.09), indicating a general positive association of acyl ghrelin with production traits, but the association weakened as time of sample collection increased. The mean ghrelin ratio tended (P = 0.08) to be positively associated with DMI in the last 35-d period. The ghrelin ratio on day 0 interacted with time of sample collection for ADG and G:F (P < 0.05), indicating an overall positive association of the ghrelin ratio with ADG and G:F. Results indicate that ghrelin is associated with DMI, ADG, and feed efficiency of finishing beef cattle, and data lend more evidence that ghrelin is involved in appetite regulation of ad libitum fed cattle.

Authors:J. De Koster; M. Hostens; K. Hermans; W. Van den Broeck; G. OpsomerPages: 117 - 126Abstract: Publication date: Available online 1 July 2016 Source:Domestic Animal Endocrinology Author(s): J. De Koster, M. Hostens, K. Hermans, W. Van den Broeck, G. Opsomer The aim of the present research was to compare different measures of insulin sensitivity in dairy cows at the end of the dry period. To do so, 10 clinically healthy dairy cows with a varying body condition score were selected. By performing hyperinsulinemic euglycemic clamp (HEC) tests, we previously demonstrated a negative association between the insulin sensitivity and insulin responsiveness of glucose metabolism and the body condition score of these animals. In the same animals, other measures of insulin sensitivity were determined and the correlation with the HEC test, which is considered as the gold standard, was calculated. Measures derived from the intravenous glucose tolerance test (IVGTT) are based on the disappearance of glucose after an intravenous glucose bolus. Glucose concentrations during the IVGTT were used to calculate the area under the curve of glucose and the clearance rate of glucose. In addition, glucose and insulin data from the IVGTT were fitted in the minimal model to derive the insulin sensitivity parameter, Si. Based on blood samples taken before the start of the IVGTT, basal concentrations of glucose, insulin, NEFA, and β-hydroxybutyrate were determined and used to calculate surrogate indices for insulin sensitivity, such as the homeostasis model of insulin resistance, the quantitative insulin sensitivity check index, the revised quantitative insulin sensitivity check index and the revised quantitative insulin sensitivity check index including β-hydroxybutyrate. Correlation analysis revealed no association between the results obtained by the HEC test and any of the surrogate indices for insulin sensitivity. For the measures derived from the IVGTT, the area under the curve for the first 60 min of the test and the Si derived from the minimal model demonstrated good correlation with the gold standard.

Authors:G. Kitahara; R. Kamata; Y. Sasaki; H. El-Sheikh Ali; S. Mido; I. Kobayashi; K. Hemmi; T. OsawaPages: 127 - 132Abstract: Publication date: October 2016 Source:Domestic Animal Endocrinology, Volume 57 Author(s): G. Kitahara, R. Kamata, Y. Sasaki, H. El-Sheikh Ali, S. Mido, I. Kobayashi, K. Hemmi, T. Osawa The aim of this study was to clarify the time-course of changes in anti-Müllerian hormone (AMH) and testosterone (T) concentrations in peripheral blood and to determine the relationships between blood AMH concentration and testicular development during the early postnatal and prepubertal periods in beef bull calves. A total of 17 Japanese Black bull calves were enrolled in this study. The wk in which the calf was born (within 6 d after birth) was defined as M 0. Blood samples were taken once in every mo from M 0 to M 6 from each bull calf, and plasma AMH and T concentrations were determined. Of the 17 calves, 10 were castrated at 6 mo of age (prepuberty) and the right testis was histologically examined. Plasma AMH concentration (means ± SE) at M 0, 1, and 2 were 123.5 ± 9.8, 189.6 ± 18.7, and 254.6 ± 14.1 ng/mL, respectively. From M 0 through M 2, plasma AMH concentration was significantly greater each mo than in the previous mo (P < 0.05); however, plasma AMH concentration significantly decreased over the last 3 mo of the study (P < 0.05). The average age at which plasma AMH concentration was the highest was 2.3 ± 0.1 mo of age. Plasma T concentration significantly increased from M 0 (0.18 ± 0.02 ng/mL) until M 6 (6.52 ± 1.41 ng/mL). Plasma AMH and T concentrations at M 4, 5, and 6 were significantly negatively correlated (P < 0.05). Linear regression did not reveal a significant relationship between Sertoli or Leydig cell numbers and plasma AMH or T concentrations, respectively. In conclusion, blood AMH concentration peaks at 2 mo of age and is negatively correlated with blood T concentration from 4 to 6 mo of age. Although prepubertal blood AMH or T concentrations did not reflect Sertoli or Leydig cell numbers at the end of the prepubertal period, blood AMH concentration may be indicative of abnormal Sertoli cells function.

Abstract: Publication date: Available online 8 December 2016 Source:Domestic Animal Endocrinology Author(s): C.J. Scudder, S.J. Niessen, B. Catchpole, R.C. Fowkes, D.B. Church, Y. Forcada Acromegaly in humans is usually sporadic, however up to 20% of familial isolated pituitary adenomas are caused by germline sequence variants of the aryl-hydrocarbon-receptor interacting protein (AIP) gene. Feline acromegaly has similarities to human acromegalic families with AIP mutations. The aim of this study was to sequence the feline AIP gene, identify sequence variants and compare the AIP gene sequence between feline acromegalic and control cats, and in acromegalic siblings. The feline AIP gene was amplified through PCR using whole-blood genomic DNA from 10 acromegalic and 10 control cats, and three sibling pairs affected by acromegaly. PCR products were sequenced and compared to the published predicted feline AIP gene. A single non-synonymous SNP was identified in exon 1 (AIP:c.9T>G) of two acromegalic cats and none of the control cats, as well as both members of one sibling pair. The region of this SNP is considered essential for the interaction of the AIP protein with its receptor. This sequence variant has not previously been reported in humans. Two additional synonymous sequence variants were identified (AIP:c.481C>T and AIP:c.826C>T). This is the first molecular study to investigate a potential genetic cause of feline acromegaly and identified a non-synonymous AIP single nucleotide polymorphism in 20 % of the acromegalic cat population evaluated, as well as in one of the sibling pairs evaluated.

Abstract: Publication date: January 2017 Source:Domestic Animal Endocrinology, Volume 58 Author(s): A. Sato, H. Ochi, Y. Harada, T. Yogo, N. Kanno, Y. Hara The purpose of this study was to investigate the expression of bone morphogenetic protein 4 (BMP4) and its receptors, bone morphogenetic protein receptor I (BMPRI) and BMPRII, in the pituitary gland of healthy adult dogs and in those with ACTH-secreting pituitary adenoma. Quantitative polymerase chain reaction analysis showed that the BMP4 messenger RNA expression level in the ACTH-secreting pituitary adenoma samples was significantly lower than that in the normal pituitary gland samples (P = 0.03). However, there were no statistically significant differences between samples with respect to the messenger RNA expression levels of the receptors BMPRIA, BMPRIB, and BMPRII. Double-immunofluorescence analysis of the normal canine pituitary showed that BMP4 was localized in the thyrotroph (51.3 ± 7.3%) and not the corticotroph cells. By contrast, BMPRII was widely expressed in the thyrotroph (19.9 ± 5.2%) and somatotroph cells (94.7 ± 3.6%) but not in the corticotroph cells (P < 0.001, thyrotroph cells vs somatotroph cells). Similarly, in ACTH-secreting pituitary adenoma, BMP4 and BMPRII were not expressed in the corticotroph cells. Moreover, the percentage of BMP4-positive cells was also significantly reduced in the thyrotroph cells of the surrounding normal pituitary tissue obtained from the resected ACTH-secreting pituitary adenoma (8.3 ± 7.9%) compared with that in normal canine pituitary (P < 0.001). BMP4 has been reported to be expressed in corticotroph cells in the human pituitary gland. Therefore, the results of this study reveal a difference in the cellular pattern of BMP4-positive staining in the pituitary gland between humans and dogs and further revealed the pattern of BMPRII-positive staining in the dog pituitary gland. These species-specific differences regarding BMP4 should be considered when using dogs as an animal model for Cushing's disease.

Abstract: Publication date: Available online 21 November 2016 Source:Domestic Animal Endocrinology Author(s): L. Li, Z. Yang, Y.-P. Zhang, S. He, X.-F. Liang, Y.-X. Tao Melanocortin-4 receptor (MC4R) plays a pivotal role in the mediation of leptin action on food intake and energy expenditure in mammals. The MC4R has also been identified in several teleosts and its importance in the regulation of fish energy homeostasis is emerging. We herein reported on the molecular cloning, tissue distribution, and pharmacological characterization of MC4R in grass carp (Ctenopharyngodon idella), an economically and ecologically important fish. We showed that grass carp MC4R (ciMC4R) consisted of a 981 bp open reading frame encoding a protein of 326 amino acids, highly homologous (>95%) to several teleost MC4Rs. Phylogenetic and synteny analysis further indicated ciMC4R was closely related to piscine MC4Rs. Using RT-PCR, we found that mc4r mRNA was expressed in the brain as well as various peripheral tissues in grass carp. The pharmacological properties of ciMC4R were investigated using four agonists, including α-melanocyte stimulating hormone (α-MSH), β-MSH, [Nle4, D-Phe7]-MSH (NDP-MSH) and adrenocorticotropic hormone (ACTH). We showed that all four ligands could bind to ciMC4R and initiate dose-dependent intracellular cAMP accumulation. Grass carp MC4R had the highest affinity for NDP-MSH. Both NDP-MSH and ACTH (1-24) exhibited higher potencies compared to the other two endogenous agonists. The ciMC4R was constitutively active, with significantly increased basal cAMP level compared with that of human MC4R (P < 0.01). The availability of ciMC4R and its pharmacological characteristics provide a basis for future investigation of its functional roles in regulating diverse physiological processes and novel insights into understanding the mechanism of food habit transition in grass carp.

Abstract: Publication date: Available online 16 November 2016 Source:Domestic Animal Endocrinology Author(s): L. Hymøller, S.K. Jensen In cattle there are 2 significant forms of vitamin D: ergocalciferol (ERG) from fungi on roughage and cholecalciferol (CHO) from vitamin supplements or endogenous synthesis in the skin. The hypothesis of the present study is that vitamin D from the 3 sources is transported in different plasma fractions in the body. This is hypothesised to explain the lower efficiency of ERG compared to CHO in securing a sufficient plasma status of 25-hydroxyvitamin D and explain the inefficient excretion of dietary CHO into milk compared to endogenous CHO. 20 vitamin D depleted cows were assigned to 5 treatments: D2 - housed indoor and fed 625 μg/d (25.000 IU) ERG; D3 - housed indoor and fed 625 μg/d CHO; D2+D3 - housed indoor and fed 625 μg/d ERG and 625 μg/d CHO; SUN - let out for daily pasture to facilitate CHO synthesis from sunlight; and D2+SUN - fed 625 μg/d ERG and let out for daily pasture. Blood samples were taken twice weekly and plasma fractionated by ultracentrifugation into 3 fractions: light lipoprotein (LLP), heavy lipoprotein (HPL), and protein and analysed for content of ERG and CHO and their liver derived metabolites 25-hydroxyergocalciferol (25ERG) and 25-hydroxycholecalciferol (25CHO), respectively. Liver biopsies were taken on the last day of the study to asses gene expression related to vitamin D metabolism. During 4 wk of study the vitamin D status in plasma increased to 19.3 to 22.8 ng/mL 25ERG in ERG treated cows with the highest concentration in D2 (P ≤ 0.05) and to 25.0 to 33.4 ng/mL 25CHO in pasture or CHO treated cows with the highest concentration in SUN (P ≤ 0.01). In plasma fractions CHO was mainly found in the HLP fraction whereas 25CHO was almost exclusively found in the protein fraction, probably due to its reported high binding affinity to vitamin D binding protein. Between 70 and 90% of 25ERG was found in the protein fraction and the remaining 25ERG in HLP, whereas ERG was found in both HLP and LLP fractions. In liver tissue the expression of vitamin D-25-hydroxylase was lower in D2+D3 (P ≤ 0.05) and SUN (P ≤ 0.05) than in the remaining groups and the vitamin D receptor (VDR) was expressed in the liver to a larger extent in D2+SUN than in D2+D3 (P ≤ 0.05) and SUN (P ≤ 0.05). In conclusion, different plasma transport mechanisms may explain the lower physiological efficiency of ERG compared to CHO in securing the vitamin D status in plasma but do not explain the lower efficiency of synthetic CHO compared to endogenous CHO from sunlight or UV light in securing a high CHO content in milk.

Abstract: Publication date: Available online 11 November 2016 Source:Domestic Animal Endocrinology Author(s): B.M. Alexander, B.C. Ingold, J.L. Young, S.R. Fensterseifer, P.J. Wechsler, K.J. Austin, D.E. Larson-Meyer Traditional confinement practices limits exposure to sunlight and Vitamin D synthesis, and vitamin insufficiency occurs even with dietary supplementation. The aim of this study was to determine the effect of limited sun exposure on serum concentration of vitamin D and the expression of vitamin D synthesizing enzymes in the liver and kidney of pigs on a vitamin D sufficient diet. White-pigmented grower pigs (29.7 ± 2.3 kg) fed 15% CP diet ad libitum providing >1200 IU vitamin D3/kg of feed were exposed to sunlight for 1 h each day at solar noon for 14 d at the spring equinox (March pigs, n=10) or summer solstice (June pigs, n=5) and again prior to slaughter in June (March pigs) and September (June pigs). Blood for the analysis of 25(OH)D was collected prior to and following sunlight exposure. Traditionally housed pigs served as controls. Following initial sun exposure, blood samples were collected from June pigs daily for 5 d and weekly for 8 wk to determine vitamin D3 and 25(OH)D decay, respectively. Kidney and liver samples were collected from the June pigs at slaughter following sun exposure for analysis of mRNA expression of vitamin D binding protein and synthesizing/degrading enzymes. ADG was not influenced (P > 0.5) by sunlight exposure. June pigs had fewer days on feed, lower (P = 0.003) ADG and were slaughtered at a lighter (P < 0.001) weight. Exposure to sunlight increased (P < 0.001) 25(OH) vitamin D for all pigs. March pigs, obtained from a Midwest producer, had lower (P < 0.001) concentration of 25(OH)D than June pigs born on-farm. Initial sunlight exposure increased serum concentration of 25(OH)D in March pigs by 200% and June pigs by 67%. Serum concentration of vitamin D3 was decreased (P < 0.05) by 72 h with 25(OH)D decreased (P < 0.05) by wk 4 following exposure. Expression of vitamin D binding protein, vitamin D synthesizing CYP2R1, CYP27A1, CYP2D25 or degrading enzyme CYP24A1 were not influenced (P ≥ 0.19) by sunlight exposure. Expression of CYP27B1 was decreased (P = 0.04) in the kidney but tended to be increased (P=0.06) in the liver following sun exposure. These results suggest limited sun-exposure can efficiently increase serum concentration of vitamin D in growing pigs with varying levels of vitamin sufficiency. The lack of major changes in vitamin synthesizing enzymes suggests the 14 d exposure period did not saturate the capacity of slaughter-weight pigs to synthesize vitamin D.

Abstract: Publication date: Available online 11 November 2016 Source:Domestic Animal Endocrinology Author(s): A.D. Vitger, B.M. Stallknecht, J.E. Miles, S.L. Hansen, A. Vegge, C.R. Bjørnvad The influence of physical activity on metabolic health in overweight dogs is unknown. This study was conducted to evaluate biomarkers of immunometabolic health in relation to changes in physical activity and adiposity. Client-owned overweight dogs participated in a 12-wk intervention based on caloric restriction combined with a training program (fitness & diet (FD) group, n = 8), or caloric restriction alone (diet-only (DO) group, n = 8). Physical activity was monitored by accelerometry. All dogs were fed the same diet and achieved similar weight loss. Fasting blood samples were collected before and after 6 and 12 wk intervention. Insulin resistance was evaluated from plasma insulin and C-peptide as well as homeostasis model assessment (HOMA-IR). Inflammation and dyslipidemia were evaluated from circulating leptin, adiponectin, C-reactive protein (CRP), monocyte chemoattractant factor-1 (MCP-1), interleukin-8 (IL-8) and cholesterol. Accelerometer counts in both groups were high compared to previous reports of physical activity in overweight dogs. No difference in blood parameters was evident between groups, evaluated by linear mixed-effects model (P > 0.05). Within the groups, the following changes were significant by t-test (P < 0.05): Leptin decreased in both groups. Within the FD group, IL-8, MCP-1 and CRP decreased at 6 wk, and IL-8 and cholesterol at 12 wk. Within the DO group, C-peptide and HOMA decreased at 6 wk and C-peptide at 12 wk. We conclude that, for both groups, weight loss resulted in minor indications of improved immunometabolic health, while this level of physical activity did not add further benefits.

Abstract: Publication date: Available online 11 November 2016 Source:Domestic Animal Endocrinology Author(s): K. Górski, T. Misztal, E. Marciniak, M.K. Zielińska-Górska, F. Fülöp, K. Romanowicz During lactation, the main surge of oxytocin is induced by a suckling stimulus. Previous studies have shown that salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline), a dopamine-derived compound, stimulates both the synthesis and the release of oxytocin in lactating sheep. The objective of the present study was to verify the hypothesis that salsolinol is involved in the mechanism that generates the oxytocin surge that occurs during suckling. Thus, a structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1MeDIQ), known to antagonize some of its actions, was infused into the third ventricle of the brain of lactating sheep nursing their offspring. Serial 30-min infusions of 1MeDIQ (4 x 60 μg/60 μl) or vehicle were administered at 30-min intervals from 10.00 to 14.00 h. The experimental period in every ewe consisted of a non-suckling period (10.00 to 12.00 h) and a suckling period (12.00 to 14.00 h). Blood samples were collected every 10 min, to measure plasma oxytocin concentration by radioimmunoassay. In control sheep, oxytocin surges of high amplitude were observed during the suckling period. The oxytocin surges induced by suckling were significantly (P < 0.01) diminished in sheep receiving 1MeDIQ infusions as compared to those that received control infusions. However, no significant effect of 1MeDIQ was observed on basal oxytocin release, before suckling. Furthermore, oxytocin release, as measured by the area under the hormone response curve (AUC), was significantly decreased by the administration of 1MeDIQ during the suckling period. This study shows that elimination of the effect of salsolinol within the central nervous system of lactating sheep attenuates the oxytocin surge induced by suckling. Therefore, salsolinol may be an important factor in the oxytocin-stimulating pathway in lactating mammals.

Abstract: Publication date: Available online 2 November 2016 Source:Domestic Animal Endocrinology Author(s): S. ThanThan, Y. Asada, T. Saito, K. Ochiiwa, H. Zhao, S.W. Naing, H. Kuwayama The present study was undertaken with the aim of examining whether and how exendin-4 (1-3) fragment, i.e., Ex-4 (1-3) fragment, contributes to the regulation of glucose. An analog of oxyntomodulin (OXM) ([Gly2, Glu3]-OXM), a glucagon analog ([Gly2, Glu3]-glucagon) and two derivatives of Ex-4 (glucandin and [Gly2, Glu3]-glucandin) were synthesized by substituting with Gly2, Glu3 at the N terminuses of OXM and glucagon and/or by attaching Ex-4 (30-39) amide at the C terminus of glucagon. Effects of these peptides on plasma insulin and glucose concentrations were investigated in cattle by conducting three in vivo experiments. In all three experiments, 0.1% BSA-saline was injected as a control. In experiment 1, glucandin (amino acid sequence was glucagon (1-29)-Ex-4 (30-39) amide) and [Gly2, Glu3]-glucandin were injected at the dose rates of 5 μg/kg body weight (BW) in 4-mo-old Holstein steers. Results showed that glucoregulatory effects of glucandin were similar to those of glucagon. [Gly2, Glu3]-glucandin stimulated insulin secretion at 2–10 min and lowered glucose concentrations at 15–75 min. Experiment 2 was carried out to better understand the glucose lowering potency of [Gly2, Glu3]-glucandin, in comparison with Ex-4 and GLP-1, using 4.5-mo-old Holstein steers. [Gly2, Glu3]-glucandin was injected at dose rates of 0.3 μg/kg BW, 1.0 μg/kg BW, 3.2 μg/kg BW and 6.4 μg/kg BW. Ex-4 and GLP-1 were injected at dose rates of 0.3 μg/kg BW. Results showed that the insulinotropic and glucose lowering effects of [Gly2, Glu3]-glucandin were not as potent as for Ex-4 and GLP-1, and the minimum effective dose of [Gly2, Glu3]-glucandin to regulate plasma glucose concentrations was 3.2 μg/kg BW. In experiment 3, [Gly2, Glu3]-OXM, and [Gly2, Glu3]-glucagon were injected at dose rates of 5 μg/kg BW in 5-mo-old Holstein steers. Both [Gly2, Glu3]-OXM and [Gly2, Glu3]-glucagon increased insulin concentration. [Gly2, Glu3]-OXM potently lowered plasma glucose, but [Gly2, Glu3]-glucagon did not change it. In summary, our findings clearly demonstrate that Ex-4 (1-3) fragment contributes to the regulation of glucose. [Gly2, Glu3]-OXM and [Gly2, Glu3]-glucandin are insulinotropic and glucose-lowering peptides. It was of interest that the substitution of the first three amino acids of OXM with Ex-4 (1-3) could reverse the up-regulation of glucose by OXM into down-regulation of glucose. In lowering glycemia, [Gly2, Glu3]-OXM seemed almost as effective as Ex-4, and [Gly2, Glu3]-glucandin was less profound than Ex-4. These findings contributed new insights into the hormonal regulation of glucose in ruminants. The action of [Gly2, Glu3]-OXM and [Gly2, Glu3]-glucandin might provide an advantage in glycemic control of insulin resistance in cattle and humans.

Abstract: Publication date: Available online 21 October 2016 Source:Domestic Animal Endocrinology Author(s): F.E. Keomanivong, A.T. Grazul-Bilska, D.A. Redmer, C.S. Bass, S.L. Kaminski, P.P. Borowicz, J.D. Kirsch, K.C. Swanson To determine the effect of feed intake and arginine treatment during different stages of the estrous cycle on pancreatic mass, digestive enzyme activity, and histological measurements, ewes (n = 120) were randomly allocated to one of three dietary groups; control (CON; 2.14 Mcal metabolizable energy/kg), underfed (UF; 0.6 x CON) or overfed (OF; 2 x CON) over 2 yr. Estrus was synchronized using a controlled internal drug release (CIDR) device for 14 d. At CIDR withdrawal, ewes from each dietary group were assigned to one of two treatments; Arg (L-Arg HCl, 155 μmol/kg BW) or Sal (approximately 10 mL Saline). Treatments were administered 3 times daily via jugular catheter and continued until slaughter on d 5 and 10 of the second estrus cycle (early luteal phase, n = 41 and mid-luteal phase, n = 39; year 1) and d 15 of the first estrus cycle (late luteal phase, n = 40; year 2. A blood sample collected from jugular catheters for serum insulin analysis before slaughter. The pancreas was then removed, trimmed of mesentery and fat, weighed, and a sample snap-frozen until enzyme analysis. Additional pancreatic samples were fixed in 10% formalin solution for histological examination of size and distribution of insulin-containing cell clusters. Data were analyzed as a completely randomized design with a factorial arrangement of treatments. Diet, treatment, and diet × treatment were blocked by year and included in the model with initial BW used as a covariate. Day of the estrous cycle was initially included in the model but later removed as no effects (P > 0.10) were observed for any pancreatic variables tested. Overfed ewes had the greatest (P < 0.001) change in BW, final BW, change in BCS, and final BCS. A diet × treatment interaction was observed for change in BW and final BW (P ≤ 0.004). Overfed and CON had increased (P < 0.001) pancreas weight (g) compared to UF ewes. Protein concentration (g/pancreas) was lowest (P < 0.001) in UF ewes while protein content (mg/kg BW) was greater (P = 0.03) in UF than OF ewes. Activity of α-amylase (U/g, kU/pancreas, U/kg of BW, and U/g protein) and trypsin (U/pancreas) was greater (P ≤ 0.003) in OF than UF ewes. Serum insulin was greatest (P < 0.001) in OF ewes. No effects were observed for pancreatic insulin-containing cell clusters. This study demonstrated that plane of nutrition affected several measurements of pancreatic function however, the dosage of Arg used did not influence pancreatic function.

Abstract: Publication date: Available online 27 October 2016 Source:Domestic Animal Endocrinology Author(s): T.Y. Ho, K.M. Rahman, M.E. Camp, A.A. Wiley, F.F. Bartol, C.A. Bagnell Nursing for 2 d from birth supports neonatal porcine uterine and cervical development. However, it is not clear how timing or duration of lactocrine signaling from birth (postnatal day = PND 0) affect development of neonatal female reproductive tract (FRT) tissues. Therefore, studies were conducted to determine effects of age at first nursing and duration of nursing from birth on specific elements of the matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) system in uterine and cervical tissues at PND 2. When nursing was initiated at 0 h or 30 min of age, targeted proteins, including proMMP9 and MMP9, were detected in uterine and cervical tissues on PND 2, as was uterine TIMP1. However, these proteins were undetectable when nursing was delayed for 12 h, and when gilts were fed milk replacer for 48 h from birth. Increasing the duration of nursing from 30 min to 12 h from birth increased uterine (P < 0.05) and cervical (P < 0.001) MMP9 levels to those observed in gilts nursed for 48 h. Similarly, uterine TIMP1 levels increased with duration of nursing. Uterine MMP2 levels were detectable but unaffected by age at first nursing or duration of nursing from birth. Uterine MMP2 and MMP9 activities, monitored by zymography, reflected immunoblotting data. Results provide evidence for the utility of MMP9 and TIMP1 as markers of age- and lactocrine-sensitive porcine female reproductive tract development.

Abstract: Publication date: Available online 27 October 2016 Source:Domestic Animal Endocrinology Author(s): O.J. Ginther, M.A.R. Siddiqui, J.M. Baldrighi, E.R. Araujo The effect of the future dominant follicle (DF), corpus luteum (CL), and side (left ovary, LO; right ovary, RO) on FSH-induced recovery (increase in diameter) of regressing subordinate follicles was studied in heifers. The DF of wave 2 and the largest subordinate follicle remained intact (controls, n = 14 heifers) or were ablated (n = 14 heifers) on a mean of 13 d postovulation when the DF was ∼ 10 mm (hour 0). Concentration of FSH (P < 0.0004) and diameter of subordinate follicles (P < 0.0002) decreased between hours −48 to 0 combined for the control and ablation groups. Thereafter, follicle diameter continued to decrease in the controls. Concentration of FSH increased (P < 0.05) and diameter of subordinates began to increase at hour 12 in the ablation group. The FSH increased to hour 24 and then returned to the hour-0 concentration by hour 72, completing the induced FSH surge. Concentration of LH began to increase at hour 0 in each group and at a similar rate between groups. Follicle recovery in the ablation group was compared among 8 subgroups as defined by the 2 sides and 4 intraovarian patterns (DF−CL pattern, both structures in same ovary; DF pattern, DF alone; CL pattern, CL alone; and devoid pattern, both structures absent). Follicle diameter increased (P < 0.05) between hours 24 and 48, and diameter at hours 24, 48, 72, and 96 involved a 3-way interaction (P < 0.0001) of pattern, side, and hour. The interaction was similar when diameter of the DF that originated from a recovered subordinate was either included or excluded in the analysis. Diameter of subordinate follicles in the ablation group at hour 96 was greater (P < 0.05) in the DF−CL/RO and DF/RO subgroups than in the devoid/LO, devoid/RO, and CL/LO subgroups. The DF−CL/LO and CL/RO subgroups were intermediate. For follicles that decreased in diameter before hour 0, a greater (P < 0.05) percentage increased after hour 0 when the ovary contained a DF and was in the RO (DF−CL/RO and DF/RO subgroups) than for the remaining subgroups even after excluding the DF that originated from a subordinate. Results supported the hypotheses that (1) an induced FSH surge can stimulate the recovery of regressing subordinate follicles and (2) recovery of regressing subordinate follicles by FSH involves an intraovarian mechanism. Our interpretation is that the intraovarian mechanism that enhances the stimulatory effect of FSH on recovery of subordinate follicles was effective only in RO and only when it contained a DF.

Authors:M.L. Johnson; D.A. Redmer; L.P. Reynolds; A.T. Grazul-BilskaAbstract: Publication date: Available online 11 October 2016 Source:Domestic Animal Endocrinology Author(s): M.L. Johnson, D.A. Redmer, L.P. Reynolds, A.T. Grazul-Bilska Gap junctions play a major role in direct, contact-dependent cell-cell communication, and they have been implicated in the regulation of cellular metabolism and the coordination of cellular functions during growth and differentiation of organs and tissues. Gap junctional channels, composed of connexin (Cx) proteins, have been detected and shown to be influenced by hormones (e.g. estrogen and /or progesterone) in uterine and placental tissues in several species. We hypothesized that 1) the mRNA for Cx26, Cx32, Cx37 and Cx43 is expressed in the uterus of ovariectomized sheep treated with estradiol-17β (E2) and in ovine placenta during early pregnancy, 2) E2-treatment of OVX ewes would cause time-specific changes in Cx26, Cx32, Cx37 and/or Cx43 mRNA expression (Experiment 1), and 3) expression of these four Cx would vary across the days of early pregnancy (Experiment 2) and will be affected by embryo origin (i.e., after application of assisted reproductive technologies [ART]; Experiment 3). Thus we collected uterine tissues at 0 to 24 h after E2 treatments (Experiment 1), and placental tissues during days 14 to 30 of early pregnancy after natural (NAT) breeding (Experiment 2) and on day 22 of early pregnancy established after transfer of embryos generated through natural breeding (NAT-ET), in vitro fertilization (IVF) or in vitro activation (IVA, parthenotes; Experiment 3). In Experiment 1, expression of Cx26, Cx37 and Cx43 mRNA increased (P < 0.05) and Cx32 mRNA decreased (P < 0.06) in both caruncular and intercaruncular tissues after E2 treatment. In Experiment 2, during early pregnancy, there were significant changes (P < 0.01) across days in expression of Cx26, Cx37 and Cx43 mRNA in the maternal placenta, accompanied by changes (P < 0.001) in Cx37 and Cx43 mRNA in the fetal placenta. In Experiment 3, in maternal placenta, Cx32 mRNA expression was decreased (P < 0.001) in NAT-ET, IVF and IVA groups compared to the NAT group; but in fetal placenta, Cx32 mRNA expression was increased (P < 0.05) in NAT-ET, IVF and IVF groups, and Cx26 mRNA expression was increased (P < 0.05) in IVA compared to NAT group. These data suggest that Cx26, Cx32, Cx37 and/or Cx43 play specific roles in E2-regulated uterine function and in placental development during early gestation both after natural mating and with application of ART.

Authors:E. Marciniak; M. Hasiec; F. Fülöp; T. MisztalAbstract: Publication date: Available online 30 September 2016 Source:Domestic Animal Endocrinology Author(s): E. Marciniak, M. Hasiec, F. Fülöp, T. Misztal This study tested the hypothesis that salsolinol, a derivative of dopamine, affects gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) secretion in lactating sheep. In the in vivo experiment, the structural analogue of salsolinol, 1-methyl-3,4-dihydroisoquinoline (1-MeDIQ), was infused into the infundibular nucleus/median eminence (IN/ME) of sheep at the 5th wk of lactation to antagonize salsolinol’s action. Simultaneously, cerebrospinal fluid from the third brain ventricle, to determine GnRH concentration, and plasma samples, to measure LH concentration, were collected. In the in vitro experiment, the anterior pituitary (AP) explants from weaned sheep were incubated in culture medium containing two doses of salsolinol, 20 and 100 μg/mL (S20 and S100, respectively). The concentration of LH in the collected media and relative expression of LHβ subunit mRNA in the AP explants were determined. No significant difference was found in mean GnRH concentration in response to 1-MeDIQ infusion, but both mean plasma LH concentration and LH pulse frequency increased significantly (p < 0.001 and p < 0.05, respectively) compared to those in controls. Significantly higher LH concentrations occurred during the 1st (p < 0.001), 2nd (p < 0.001), and 4th (p < 0.05) h of 1-MeDIQ infusion. In the in vitro study, both the S20 and S100 doses of salsolinol caused a significant decrease in the mean medium LH concentration compared to that in the control (p < 0.01 and p < 0.001, respectively). Salsolinol had no effect on the relative LHβ subunit mRNA expression in the incubated tissue. In conclusion, salsolinol is a potential inhibitor of the secretory activity of the gonadotropic axis in lactating sheep, at least at the AP level. Although no significant changes in GnRH release were directly confirmed, an increase in the frequency of LH pulses, does not allow to exclude the central action of salsolinol.

Authors:C.K. Hughes; M.M. Xie; S.R. McCoski; A.D. EalyAbstract: Publication date: Available online 13 September 2016 Source:Domestic Animal Endocrinology Author(s): C.K. Hughes, M.M. Xie, S.R. McCoski, A.D. Ealy Leptin is involved in various reproductive processes in humans and rodents, including placental development and function. The specific ways that leptin influences placental development and function in cattle are poorly understood. This work was completed to explore how leptin regulates hormone, cytokine and metalloprotease transcript abundance and cell proliferation in cultured bovine trophoblast cells. In the first set of studies, cells were cultured in the presence of graded recombinant bovine leptin concentrations (0, 10, 50, 250 ng/mL) for 6- or 24-h. Transcript profiles were examined from extracted RNA. Leptin supplementation did not affect abundance of the maternal recognition of pregnancy factor, interferon-tau (IFNT), but leptin increased (P < 0.05) abundance of chorionic somatomammotropin hormone 2 (CSH2; i.e. placental lactogen) at both 6- and 24-h at each concentration tested. At 24-h, the greatest CSH2 abundance (P < 0.05) was detected in cells supplemented with 50 ng/mL leptin. Transcript abundance of the remodeling factor, metalloprotease 2 (MMP2), was greater (P < 0.05) in leptin-treated cells at 24-h but not at 6-h. The 24-h MMP2 response was greatest (P < 0.05) at 250 ng/mL. Transcript abundance for MMP9 was not altered by leptin treatment. In a separate set of studies, cell proliferation assays were completed. Leptin supplementation did not affect CT1 proliferation at any dose tested. In conclusion, leptin supplementation did not affect bovine trophoblast cell proliferation or IFNT expression, but leptin increases CSH2 and MMP2 transcript abundance. Both of these factors are involved with peri- and post-implantation placental development and function, and this implicates leptin as a potential mediator of early placental development and function in cattle.

Authors:E. Kamanga-Sollo; K.J. Thornton; M.E. White; W.R. DaytonAbstract: Publication date: Available online 13 September 2016 Source:Domestic Animal Endocrinology Author(s): E. Kamanga-Sollo, K.J. Thornton, M.E. White, W.R. Dayton In feedlot steers, estradiol-17β (E2) and combined E2 and trenbolone acetate (TBA) (a testosterone analog) implants enhance rate and efficiency of muscle growth; and, consequently, these compounds are widely used as growth promoters in several countries. Treatment with E2 stimulates protein synthesis rate and suppresses protein degradation rate in fused bovine satellite cell (BSC) cultures, however the mechanisms involved in these effects are not known with certainty. Although the genomic effects of E2 mediated through the classical estrogen receptors have been characterized, recent studies indicate that binding of E2 to the G protein-coupled estrogen receptor (GPER)-1 mediates non-genomic effects of E2 on cellular function. Our current data show that inhibition of G protein-coupled estrogen receptor (GPER)-1, matrix metalloproteinases 2 and 9 (MMP2/9), or heparin binding epidermal growth factor-like growth factor (hbEGF) suppresses E2-stimulate protein synthesis rate in cultures bovine satellite cells (BSC) (P < 0.001) suggesting that all of these are required in order for E2 to stimulate protein synthesis in these cultures. In contrast, inhibition of GPER-1, MMP2/9, or hbEGF has no effect on the ability of E2 to suppress protein degradation rates in fused BSC cultures indicating that these factors are not required in order for E2 to suppress protein degradation rate in these cells. Furthermore, treatment of fused BSC cultures with E2 increased (P < 0.05) pAKT levels in fused BSC cultures compared to untreated control cultures, indicating that the pAKT pathway may play a role in E2-stimulated effects on cultured BSC. In summary, our current data show that active GPER-1, MMP2/9, and hbEGF are necessary for E2-stimulated protein synthesis but not for E2-simulated suppression of protein degradation in cultured BSC. Additionally, E2-treatment increases pAKT levels in cultured BSC.

Authors:Kalbe Block; Lefaucheur K.-P. Bellmann Pfuhl Puppe Otten C.C. MetgesAbstract: Publication date: Available online 16 August 2016 Source:Domestic Animal Endocrinology Author(s): C. Kalbe, D. Lösel, J. Block, L. Lefaucheur, K.-P. Brüssow, O. Bellmann, R. Pfuhl, B. Puppe, W. Otten, C.C. Metges, C. Rehfeldt The aim of our study was to characterize the immediate phenotypic and adaptive regulatory responses of fetuses to different in utero conditions reflecting inadequate maternal protein supply during gestation. The gilts fed high (250% above control) or low (50% under control) protein diets isoenergetically adjusted at the expense of carbohydrates from the day of insemination until the fetuses were collected at d 64 or 94 of gestation. We analyzed body composition, histo-morphology, biochemistry, and mRNA expression of fetal skeletal muscle. Both diets had only marginal effects on body composition and muscular cellularity of fetuses including an unchanged total number of myofibers. However, mRNA expression of myogenic regulatory factors (MYOG, MRF4, P ≤ 0.1), IGF system (IGF1, IGF1R, P ≤ 0.05) and myostatin antagonist FST (P = 0.6, in males only) was reduced in the fetal muscle exposed to a maternal low protein diet. As a result of excess protein, MYOD, MYOG, IGF1R and IGFBP5 mRNA expression (P ≤ 0.05) was upregulated in fetal muscle. Differences in muscular mRNA expression indicate in utero regulatory adaptive responses to maternal diet. Modulation of gene expression immediately contributes to the maintenance of an appropriate fetal phenotype that would be similar to that observed in the control fetuses. Moreover, we suggest that the modified gene expression in fetal skeletal muscle can be viewed as the origin of developmental muscular plasticity involved in the concept of fetal programming.

Authors:F.X. Donadeu; IoannidisAbstract: Publication date: Available online 13 August 2016 Source:Domestic Animal Endocrinology Author(s): F.X. Donadeu, J. Ioannidis In a previous microarray study we identified a subset of miRNAs which expression was distinctly higher in atretic than healthy follicles of cattle. In the present study we investigated the involvement of those miRNAs in granulosa and theca cells during atresia. RT-qPCR confirmed that miR-21-5p/-3p, miR-150, miR-409a, miR-142-5p, miR-378, miR-222, miR-155 and miR-199a-5p were expressed at higher levels in atretic than healthy follicles (9-17 mm, classified based on steroidogenic capacity). All miRNAs except miR-21-3p and miR-378 were expressed at higher levels in theca than granulosa cells. The expression of 13 predicted miRNA targets was determined in follicular cells by RT-qPCR, revealing downregulation of HIF1A, ETS1, JAG1, VEGFA and MSH2 in either or both cell types during atresia. Based on increases in miRNA levels simultaneous with decreases in target levels in follicular cells, several predicted miRNA-target interactions were confirmed that are putatively involved in follicular atresia, namely miR-199a-5p/miR-155-HIF1A in granulosa cells, miR-155/miR-222-ETS1 in theca cells, miR-199a-5p-JAG1 in theca cells, miR-199a-5p/miR-150/miR-378-VEGFA in granulosa and theca cells, and miR-155-MSH2 in theca cells. These results offer novel insight on the involvement of miRNAs in follicle development by identifying a miRNA-target network that is putatively involved in follicle atresia.

Authors:Michele Plewes; Patrick Burns Peter Graham Richard Hyslop George BarisasAbstract: Publication date: Available online 12 August 2016 Source:Domestic Animal Endocrinology Author(s): Michele R. Plewes, Patrick D. Burns, Peter E. Graham, Richard M. Hyslop, B. George Barisas Lipid microdomains are ordered regions on the plasma membrane of cells, rich in cholesterol and sphingolipids, ranging in size from 10 to 200 nm in diameter. These lipid-ordered domains may serve as platforms to facilitate co-localization of intracellular signaling proteins during agonist-induced signal transduction. It is hypothesized that fish oil will disrupt the lipid microdomains, increasing spatial distribution of these lipid-ordered domains and lateral mobility of the prostaglandin (PG) F2α (FP) receptors in bovine luteal cells. The objectives of this study were to examine the effects of fish oil on 1) the spatial distribution of lipid microdomains, 2) lateral mobility of FP receptors and 3) lateral mobility of FP receptors in the presence of PGF2α on the plasma membrane of bovine luteal cells in vitro. Bovine ovaries were obtained from a local abattoir and corpora lutea were digested using collagenase. In Experiment 1, lipid microdomains were labeled using cholera toxin subunit B Alexa Fluor 555. Domains were detected as distinct patches on the plasma membrane of mixed luteal cells. Fish oil treatment decreased fluorescent intensity in a dose dependent manner (P < 0.01). In Experiment 2, single particle tracking was used to examine the effects of fish oil treatment on lateral mobility of FP receptors. Fish oil treatment increased micro- and macro-diffusion coefficients of FP receptors as compared to control cells (P < 0.05). In addition, compartment diameters of domains were larger and residence times were reduced for receptors in fish oil treated cells (P < 0.05). In Experiment 3, single particle tracking was used to determine the effects of PGF2α on lateral mobility of FP receptors and influence of fish oil treatment. Lateral mobility of receptors was decreased within 5 min following addition of ligand for control cells (P < 0.05). However, lateral mobility of receptors was unaffected by addition of ligand for fish oil treated cells (P > 0.10). The data presented provide strong evidence that fish oil causes a disruption in lipid microdomains and affects lateral mobility of FP receptors in the absence and presence of PGF2α.

Authors:Chang Frandsen; J.E. GadsbyAbstract: Publication date: Available online 15 July 2016 Source:Domestic Animal Endocrinology Author(s): J. Chang, S. Frandsen, J.E. Gadsby The porcine corpus luteum (CL) displays delayed sensitivity to PGF-2α (luteolytic sensitivity, [LS]) until days 12 to 13 of cycle. The control of LS is unknown, but it is temporally associated with macrophage (which secrete TNF-α) infiltration into the CL. Other studies showed that TNF-α induces LS in vitro and that prostaglandins may be involved in this mechanism. In experiment 1, PGF-2α and PGE secretion by luteal cells (LCs) was measured on days 4 to 14 of the estrous cycle, and the expression of PTGFS/AKR1B1 and PTGES/mPGES-1, by Western blot, before (day 7) vs after (day 13) the onset of LS. Results showed that the PGF-2α:PGE ratio increased significantly (P < 0.05) from day 4 to 13–14, and PTGFS/AKR1B1 and PTGES/mPGES-1 were significantly increased (P < 0.05) on day 13 (vs day 7). In experiment 2, LCs were collected from porcine CL at early (∼days 4–6) or mid (∼days 7–12) stages of the estrous cycle and cultured with 0, 0.1, 1, or 10-ng/mL TNF-α. Results showed that TNF-α significantly increased (P < 0.05) messenger RNA (mRNA) expression of cyclooxygenase (COX)-2 and mPGES-1 but not AKR1B1. TNF-α had no significant effects on AKR1B1 or mPGES protein abundance. TNF-α significantly increased (P < 0.05) PGE-2 but had no effect on PGF2αs secretion or on the PGF2α:PGE2 ratio. In conclusion, although TNF-α increased COX2 and mPGES-1 mRNA, and PGE-2 secretion in vitro, it did not increase the PGF2α:PGE2 ratio. Studies are currently directed toward exploring other pathways (eg, FP receptor signaling) by which TNF-α induces LS in the porcine CL.

Authors:C.B. Steinhauser; F.W. Bazer R.C. Burghardt G.A. JohnsonAbstract: Publication date: Available online 15 July 2016 Source:Domestic Animal Endocrinology Author(s): C.B. Steinhauser, F.W. Bazer, R.C. Burghardt, G.A. Johnson Progesterone (P4) stimulates production and secretion of histotroph, a mixture of hormones, growth factors, nutrients, and other substances required for growth and development of the conceptus (embryo/fetus and placental membranes). Progesterone acts through the progesterone receptor (PGR); however, there is a gap in our understanding of P4 during pregnancy because PGR have not been localized in the uteri and placentae of pigs beyond day 18. Therefore, we determined endometrial expression of PGR messenger RNA (mRNA) and localized PGR protein in uterine/placental tissues throughout the estrous cycle and through day 85 of pregnancy in pigs. Further, 2 components of histotroph, tartrate-resistant acid phosphatase 5 (ACP5; uteroferrin) and secreted phosphoprotein 1 (SPP1; osteopontin) proteins, were localized in relation to PGR during pregnancy. Endometrial expression of PGR mRNA was highest at day 5 of the estrous cycle, decreased between days 5 and 11 of both the estrous cycle and pregnancy, and then increased between days 11 and 17 of the estrous cycle (P < 0.01), but decreased from days 13 to 40 of pregnancy (P < 0.01). Progesterone receptor protein localized to uterine stroma and myometrium throughout all days of the estrous cycle and pregnancy. PGR were expressed by uterine luminal epithelium (LE) between days 5 and 11 of the estrous cycle and pregnancy, then PGR became undetectable in LE through day 85 of pregnancy. During the estrous cycle, PGR were downregulated in LE between days 11 and 15, but expression returned to LE on day 17. All uterine glandular epithelial (GE) cells expressed PGR from days 5 to 11 of the estrous cycle and pregnancy, but expression decreased in the superficial GE by day 12. Expression of PGR in GE continued to decrease between days 25 and 85 of pregnancy; however, a few glands near the myometrium and in close proximity to areolae maintained expression of PGR protein. Acid phosphatase 5 protein was detected in the GE from days 12 to 85 of gestation and in areolae. Secreted phosphoprotein 1 protein was detected in uterine LE in apposition to interareolar, but not areolar areas of the chorioallantois on all days examined, and in uterine GE between days 35 and 85 of gestation. Interestingly, uterine GE cells adjacent to areolae expressed PGR, but not ACP5 or SPP1, suggesting these are excretory ducts involved in the passage, but not secretion, of histotroph into the areolar lumen and highlighting that P4 does not stimulate histotroph production in epithelial cells that express PGR.

Authors:BaloghAbstract: Publication date: Available online 21 July 2016 Source:Domestic Animal Endocrinology Author(s): J. Thuróczy, J. Szilágyi, L. Müller, L. Balogh Thyroxine (T4) and triiodothyronine (T3) concentrations in pregnant and non-pregnant bitches were measured. The allantoic and amniotic fluid samples were collected separately in the third week of pregnancy and fetal blood samples were collected in the fourth week of pregnancy. There was no difference between T4 results in the pregnant and non-pregnant animals, but the measured serum concentrations exceeded the healthy range for normal adults. Serum T4 concentrations were lower in the fetus than in adults (P < 0.01). Fetal T4 concentrations continuously increased and reached 13.38 ± 6.19 nmol/L before birth. The fetal serum T4 concentrations were lower than the T4 concentrations in allantoic and amniotic fluid until the seventh week and the fetal serum T3 concentrations were lower than in fetal fluids throughout the pregnancy (P < 0.01). Maximum T3 concentrations in allantoic and amniotic fluid exceeded the concentrations in the fetal and maternal serum. It is conceivable that the considerable differences between maternal and fetal serum T4 concentrations in healthy animals are explained by the T4 impermeability of the placenta. Extremely high maternal T4 (193.5 nmol/L) in one bitch was associated with T4 concentrations under the detection limit in the fetal fluids and serum suggesting an inhibitory effect. The T4 concentrations in all of the fetal fluids and serum were under the detectable concentration that can be defined by 3.0 nmol/L in that bitch. We have demonstrated that fetal thyroid glands start functioning independently at the same time as thyroid cell formation in the dog, but the overproduction of maternal T4 may have a suppressive effect on fetal iodothyronine production.

Authors:B.M. Socha; A.A. A.J. KorzekwaAbstract: Publication date: Available online 25 July 2016 Source:Domestic Animal Endocrinology Author(s): M. Łupicka, B.M. Socha, A.A. Szczepańska, A.J. Korzekwa Adenomyosis is uterine dysfunction defined as the presence of endometrial glands within the myometrium. It is suggested that adenomyosis is oestrogen-dependent pathology, and prolactin (PRL) also affects its development. In the uterus of ruminants, PRL stimulates gland proliferation and function. We hypothesised that in the bovine uterus, expression of PRL and its receptors (PRLRs) during adenomyosis is disturbed and modulated by oestradiol (E2). Uterine tissues were collected post mortem from cows; epithelial, stromal and myometrial cells were isolated; and cultured and treated with E2. Material was divided into two groups: control (non-adenomyotic) and uteri with adenomyosis. In adenomyotic uterine tissue, PRL and its long-form receptor (lPRLR) protein were increased, as determined by western blotting. Immunohistostaining showed that during adenomyosis, PRL and its receptors are highly expressed in adenomyotic lesions. In cultured myometrial cells, protein expression of PRL and its receptors was increased during adenomyosis. Oestradiol decreased PRLRs protein expression in non-adenomyotic stromal cells and in adenomyotic myometrial cells, and increased PRL secretion by adenomyotic myometrial cells. Moreover, PRL secretion was increased in untreated epithelial and stromal cells during adenomyosis. On the other hand, in stromal cells, PRLRs mRNA and protein expression was decreased, as determined by real-time PCR and western blotting, respectively. Obtained results show that significant changes in PRL and PRLRs expression are observed in uterine tissue and cells during adenomyosis, which were also affected by E2. These data suggest involvement of PRL in adenomyosis development and the link between PRL and E2 actions during the dysfunction in cows.

Authors:Lim SongAbstract: Publication date: October 2016 Source:Domestic Animal Endocrinology, Volume 57 Author(s): W. Lim, G. Song Tyrosine aminotransferase (TAT) catalyzes the transamination of tyrosine to p-hydroxyphenylpyruvate. Accumulation of tyrosine in the body due to a genetic mutation in the TAT gene causes tyrosomia type II in humans. The TAT gene is regarded as a model for studying steroid-inducible factors regulating a variety of biological functions of TAT. However, little is known of the effects of estrogen on the expression of the TAT gene in chickens. Therefore, in the present study, we identified expression of the avian TAT gene in various organs. The results showed the TAT was detected predominantly in the liver and reproductive organs including testis, oviduct, and ovary. Specifically, TAT mRNA was expressed abundantly in the glandular and luminal epithelia of the oviducts in response to endogenous and exogenous estrogens which also induce dramatic morphological changes in the oviduct of chickens. In addition, target microRNAs of TAT (miR-1460, miR-1626-3p, miR-1690-5p, and miR-7442-3p) were found to modulate expression of the TAT gene. Especially, miR-1690-5p influenced TAT gene transcription by binding directly to its 3′-UTR region. Moreover, the expression of TAT was abundant in glandular epithelia of cancerous but not normal ovaries from laying hens. Taken together, our findings suggest that TAT plays an important role in the cytodifferentiation of oviducts in response to estrogen and in the progression of ovarian cancer in chickens.